David Cameron said there would be a "monumental battle" to keep the UK together.
Mr Cameron used his speech to the Scottish Conservative Party conference in Edinburgh to warn against a Yes vote in the poll.
He said: "Six months from now the day will dawn , the polling booths will open, the voters will come out and the people of Scotland will decide - stay or go.
"Stick with the UK or walk away.
"If the Scottish people vote Yes in September, then Scotland will become an independent country, there will be no going back, no second chances."
He added: "We face a monumental battle to keep our United Kingdom together."
The Prime Minister, who was introduced by Scottish party leader Ruth Davidson, said a No vote "can" lead to greater powers at Holyrood with Scotland still in the UK.
He said offering more responsibility is not a consolation prize for First Minister Alex Salmond.
"Let me be absolutely clear: a vote for 'no' is not a vote for 'no change'," he said.
"We are committed to making devolution work better still.
"Not because we want to give Alex Salmond a consolation prize if Scotland votes No, but because it's the right thing to do.
"Giving the Scottish Parliament greater responsibility for raising more of the money it spends - that's what Ruth believes, and I believe it too.
"So here's the re-cap: vote yes, that is total separation.
"Vote no, that can mean further devolution more power to the Scottish people and their Parliament, but with the crucial insurance policy that comes with being part of the UK."
Mr Cameron, speaking at the Edinburgh International Conference Centre, praised the "spirit" of the Scottish party, which has just one MP at Westminster.
"That spirit is about fighting through adversity," he said.
"It's about having a stout heart, an unwavering gaze, and, let's be frank, having a skin as thick and impenetrable as the SNP's White Paper on independence.
His opponents in the Yes Scotland campaign for independence argue that Mr Cameron's Government does not reflect Scottish "values".
Mr Cameron told the conference: "Our values are Scotland's values.
"We've got to make this case fearlessly, passionately, and that is exactly what our brilliant leader Ruth is doing today."
He drew on recent interventions from major businesses, such as BP and Shell, in the referendum debate.
And he vowed to tackle accusations of "scaremongering" over the country's future, saying he wants to "take that myth apart".
He repeated the warning that the UK Government would not agree to a formal currency union to allow an independent Scotland to keep sterling.
"There are a few myths doing the rounds," he said.
"There's the myth that any talk about the consequences of separation is all bluff and bluster, or even bullying. Warnings on the currency. Warnings on the EU.
"The Nationalists say this is a big political conspiracy from south of the border, just ignore it. But that is wrong and, frankly, irresponsible."
He defended interventions from the president of the European Commission, BP, Shell, RBS and Barclays.
"These are not political puppets, they are serious, non-partisan figures," he said.
"So the idea that these are empty warnings and political scaremongering is a myth, and we owe it to the people of Scotland to take that myth apart."
The Prime Minister went on to emphasise the UK's shared history, values, identity and culture, touching on events such as the First World War, the Olympics and the Commonwealth Games.
Remaining part of the UK would also provide many benefits for industry, he told delegates.
Mr Cameron also argued that the Coalition's "serious long-term economic plan" for the UK was working in Scotland.
He said: "We have seen six consecutive quarters of Scottish growth. Employment is up by over 100,000 people since the election."
Meanwhile "2.2 million Scottish taxpayers have more money in their pockets thanks to our tax cuts", he said.
"So together we've come through the great recession as the fastest-growing economy in Europe and together there are huge prizes to be taken in the future."
The Prime Minister continued: "In oil and gas - if we maximise this asset our economy could be boosted by £200 billion over the next 20 years.
"And, yes, that's about the bravery of those who work in deep-sea exploration and the experts in Aberdeen, but it's also about encouraging investment with the proven, long-term stability of the UK economy - the broad shoulders that make it all possible."
He added: "We have centuries of experience in shipbuilding on the Clyde, but that's backed up by the UK defence budget - in the top five on the planet."
These industries, Mr Cameron said, are "better off" as part of the UK.
Having a "place around the top table" at European and international organisations also ensures the best trade deals, he told delegates.
"And I always make sure personally that Scottish-based companies are there with me at the top table.
"Like Aggreko, headquartered in Glasgow. I've taken them on trade missions to Brazil, India, South East Asia, shaking hands and doing deals that mean more work and more wealth right here in Scotland.
"Our place in the world matters - and the fact is we matter more as a United Kingdom."
The nations within the UK are "a family", the Prime Minister concluded.
"We're there for each other," he said.
"When you look around the world, so often, tragically, neighbourliness has been lost and replaced by wars and feuds.
"We don't do that. We don't slam the doors and turn compatriots into foreigners. We work together."